Project Summary This project focuses on chloroplasts as central player in the generation of immune signals and the regulation of programmed cell death (PCD) required for innate immune responses against pathogen infection. Although mitochondria play a central role during mammalian PCD, emerging evidence suggests that in plants chloroplasts have a critical function in executing localized PCD that limits pathogen spread. Chloroplasts in addition to being involved in the generation of immune signals such as reactive oxygen species (ROS) and defense hormone salicylic acid (SA), also participate directly in the recognition of pathogen. Interestingly, the chloroplasts dynamically change their morphology during immune responses and send out tubular projections called stromules. These induced stromules use the cytoskeleton to extend and then anchor to the nucleus, which facilitate perinuclear clustering of chloroplasts and transport chloroplast-generated ROS and defense proteins to the nucleus. The overall goal of this application is to use combination of novel cell biology, genetics, proteomics and computational approaches to unravel the mechanistic basis of stromule driven periunclear chloroplast clustering and determine the identity and function of immune signals released from chloroplasts to nuclei. Specifically, Aim 1 will examine organelle and cytoskeleton dynamics during plant innate immunity. Novel computational methods quantifying stromule features and dynamics will be further developed and applied to understand chloroplasts, their stromules, and cytoskeleton dynamics during innate immunity. Both plant and animal pathogens target the cytoskeleton, and this aim will examine how pathogen effectors alter the cytoskeleton and related organelle dynamics as a virulence strategy. Aim 2 will investigate immune signals required for stromule induction and the release of chloroplast signals. The relationship of different ROS sources, organelle movement and PCD during immune responses will be examined using a combination of genetic tools and the computational methods developed in Aim 1. In Aim 3, known chloroplast-derived immune signals will be characterized and novel protein signals will be identified. The downstream targets of H2O2 in the nucleus will be elucidated and studied. Mass spectrometry will be used to identify proteins that are transported from chloroplasts to nuclei. Genetic approaches will be employed to examine their role during PCD. Lastly, the mechanism of release from chloroplasts during innate immunity will be studied to determine if it is akin to mitochondrial release during apoptosis in mammals. Understanding the role of different organelles during PCD and innate immunity will provide a unified mechanistic basis of cell death and cell survival process that occur in response to infectious pathogens. The results from our model systems will impact broadly on understanding of organelle-to-nuclear communication that influence innate immunity against infectious diseases.